Glycated proteins are contained in biological samples such as body fluid and hair, and body fluid includes blood, and such. The concentration of glycated proteins present in the blood depends on the concentration of sugars such as glucose dissolved in the serum, and recently, in the field of clinical diagnosis, measurement of the concentration of hemoglobin A1c (Non-Patent Document 1), which is a glycated protein in the blood, is being used to diagnose and monitor diabetes mellitus. As methods for measuring this hemoglobin A1c, instrumental analytical methods using high performance liquid chromatography (HPLC) (Non-Patent Document 2), immunoassays using antigen-antibody reactions (for example, Non-Patent Document 3), and such had been known, but in recent years, enzymatic assays have been developed, and for example, a method using a protease and a fructosyl peptide oxidase (Patent Document 1) has been developed. Enzymatic assays can be applied to versatile automated analyzers, and since the operations are also simple, their development is increasing.
The fructosyl peptide oxidase used in enzymatic assays is an enzyme that catalyzes the reaction which produces a sugar osone (an α-keto aldehyde), a peptide, and hydrogen peroxide by oxidative cleaving, in the presence of oxygen molecules, the C—N bond in the ketose derivative produced by Amadori rearrangement of glucosylamine produced by the reaction between the hemiacetal of glucose and the N-terminal amino group of a peptide.
In the case of enzymatic assays, as shown in FIG. 1, a method is known in which hemoglobin A1c is first degraded with a protease, and α-fructosyl valyl histidine (hereinafter, denoted as α-FVH) is produced from the N terminus of the β-chain of hemoglobin; next, fructosyl peptide oxidase is made to act on the produced α-FVH, hydrogen peroxide which is produced is applied to oxidative condensation in the presence of peroxidase to afford a quinone dye, and the produced amount is determined by colorimetry using a spectrophotometer (Patent Document 1).
However, ε-fructosyl lysine and glycated peptides containing it form as byproducts by protease treatment, and it has been pointed out that there is a risk that, when fructosyl peptide oxidase acts on them, the measured values of hemoglobin A1c may be higher than the true values (Patent Document 2).
Fructosyl peptide oxidase has been found from bacteria, fungi, and plants. For example, fructosyl peptide oxidase derived from the genus Achaetomiella, the genus Chaetomium (Patent Document 3), the genus Curvularia (Patent Document 2), the Rosaceae family, the Vitaceae family, the Apiaceae family (Patent Document 4), the Zingiberaceae family (Patent Document 5), and such are known.
However, fructosyl peptide oxidases reported so far had drawbacks, such as:
(1) the activity towards an α-glycated dipeptide (α-fructosyl valyl histidine) in comparison to an α-glycated amino acid (for example, α-fructosyl valine) is not necessarily high;
(2) as described above, in addition to the N-terminal α-glycated dipeptide, it also acts on an ε-glycated amino acid in which a sugar is bound to the ε-amino group of lysine (ε-fructosyl lysine), and increases the measured values in hemoglobin A1c measurements; and
(3) in the case of measurement methods using enzymes, the enzymes become unstable during measurement or storage.
To overcome these drawbacks, enzymes with decreased reactivity towards ε-fructosyl lysine as a result of artificial introduction of mutations into fructosyl peptide oxidase (Patent Document 4), enzymes with increased heat resistance also due to introduction of mutations (Non-Patent Document 4), and such have been reported. However, the existence of enzymes that have simultaneously overcome the above-mentioned drawbacks of (1) to (3) at a high level is still not known.    [Non-Patent Document 1] Clinical Chemistry and Laboratory Medicine Vol. 36, p. 299-308 (1998).    [Non-Patent Document 2] Chromatogr. Sci., Vol. 10, p. 659 (1979).    [Non-Patent Document 3] Nihon Rinsho Kensa Jidoka Gakkai Kaishi (Journal of the Japan Society for Clinical Laboratory Automation), Vol. 18, No. 4, p. 620 (1993).    [Non-Patent Document 4] Appl. Microbiol. Biotechnol., Vol. 78, No. 5, p. 775-781 (2008).    [Patent Document 1] Japanese Patent Application Kokai Publication No. (JP-A) 2001-95598 (unexamined, published Japanese patent application).    [Patent Document 2] International Publication No. WO 2004/104203 pamphlet.    [Patent Document 3] JP-A (Kokai) 2003-235585.    [Patent Document 4] International Publication No. WO 2004/038033 pamphlet.    [Patent Document 5] International Publication No. WO 2004/038034 pamphlet.